The present study establishes that it is indeed possible to differentiate a significant number of DA neurons from several different hES cell lines, including the previously unstudied HUES7and 8 lines, both in vitro and in vivo, using a protocol of defined human-derived reagents in a simple serum-free media. According to this procedure, hES cells will differentiate into β-tubIII+ neurons, many of which express DA traits such as TH, AADC, Ptx-3, Lmx-1b, Nurr-1 and DAT after only three weeks in culture with several chemically-defined media additives and substrates, all of human derivation. Alternatively, cells harvested from culture at earlier stages in the protocol can continue the DA differentiation process in vivo, expressing differentiated DA traits within several weeks of transplantation into the brain.
This protocol differs from previously published procedures in a number of important ways. In earlier studies, DA differentiation of hES cells required incubation in culture with other cell types (PA6 mouse stromal line; human HepG2 liver line) or in cell-CM, usually containing fetal calf serum and/or other undefined components (4
). In several recent studies, efforts have been made to move to a serum-free defined protocol for neuronal (9
) or DA differentiation (19
) of hES cells. With only one exception (19
), however, media was supplemented with serum substitutes, such as B27, and often cells were grown on Matrigel, both of which contain undefined proprietary components and growth substances of animal origin. Only in the case where hES cells were grown as spheres in suspension was a simple unsupplemented serum-free media sufficient to support the differentiation of DA neurons (19
), possibly due to the local production of necessary factors in the spheres. However, DA neurons differentiated in this manner did not survive in adherent culture and only rarely survived engraftment in vivo (19
). Importantly, neural progenitor cells, also present in these spheres, did not go on to develop into DA neurons after transplantation. The current protocol likewise employs a relatively simple serum-free media but maintains cells for the most part on adherent substrates. Unlike hES suspension cultures, these attached cells did not develop beyond the neural progenitor stage to produce DA neurons until further differentiation with dbcAMP in culture or transplantation in vivo where some cells carried on the DA differentiation process. Interestingly, mouse ES-derived progenitors sharing similar DA potentiality in the graft have also recently been reported (8
). Whether these cells represent those which express DA fate determinant genes (2
) and are normally committed to a DA fate in the ventral midbrain remains to be determined. Regardless, the findings of this study, unlike those of previous hES studies, establish that a simple media containing several defined human additives can foster the DA developmental potential of hES-derived progenitor cells. Studies to determine the long-term survival and function of these transplanted hES-derived progenitors cells are still ongoing.
It will be important to develop feeder-free support systems not only for the propagation but also the initial establishment of undifferentiated ES cells so that exposure of human cells to mouse antigens can be entirely eliminated. Although a number of feeder-free systems have been described (1
), all hES lines to date have been established and repeatedly passaged on mouse feeders prior to transfer to a feeder free-system for cell propagation. Thus far, the feeder free protocols which we have tried (3
), have not been compatible with the DA differentiation of hES cell lines developed in this protocol. Finally, it is essential that the bovine serum albumin and porcine transferrin found in KOSR basal media be replaced with human products that can support hES development; currently our efforts (unpublished findings) and those of Gibco (personal communication) in this regard have not been successful. Advances such as these however are critical for the translation of these technologies from the bench to the clinical treatment of patients with diseases such as Parkinson’s.